Vitreous cryopreservation and thawing of adipose-derived stem cells/demineralized bone matrix / 中国组织工程研究

ABSTRACT

BACKGROUND: Short-term (1 week) vitreous cryopreservation avoiding the formation of ice crystals has been achieved in preserving tissue-engineered bone composed of adipose-derived stem cells (ADSCs)/demineralized bone matrix (DBM) compound through adjusting particular composition of cryopreservation fluid. However, whether vitreous cryopreservation can be utilized to cryopreserve tissue-engineered bone for long term (12 weeks) and maintain cellular viability and osteogenic function after rewarming remains unclear. OBJECTIVE: To investigate the effects of vitreous cryopreservation on viability and osteogenic function of ADSCs for short-term (1 week) and long-term (12 weeks) cryopreservation. METHODS: ADSCs were isolated from New Zealand rabbits and expended to passage 3. Cells at passage 3 were seeded onto DBM derived from porcine trabecular bone and followed by 1 week osteogenic induction. The tissue-engineered bone was transferred to freezing vials of 2 mL containing vitreous cryopreservation fluid and then directly quenched into liquid nitrogen. The composition of cryopreservation fluid was 30% dimethyl sulfoxide, 70% low glucose-Dulbecco's modified Eagle medium (L-DMEM), 0.8 mol/L trehalose. Following vitrification for 1 week or 12 weeks, the composite of ADSCs/DBM was removed and thawed. After rewarming, ADSCs viability were viewed under confocal laser microscope by staining viable cells with the green fluorescent dye Calcein AM and the red fluorescent dye Propidium iodide at days 1, 3, 7, 11 and 13. The number of cells seeded onto the DBM was assayed by Hochest33258 at days 1, 3, 5, 7, 9, 11 and 13. Meanwhile, alkaline phosphatase (ALP) activity was also assayed by PNP microplate method at days 1, 4, 7, 10, 14 and 21. Osteogenic gene expression including Runx2, OCN, ALP, COL-1 was detected by real- time PCR at days 1, 4, 7, 10, 14 and 21. RESULTS AND CONCLUSION: After cryopreservation of 1 week or 12 weeks, it was found that more red-staining live cells was observed at 1 day post-rewarming by live/dead double staining, and the green-staining live cells increased at 3 days. By Hoechst 33258 assay, it was found that the cell number decreased at 1 and 3 days post-rewarming, compared with pre-cryopreservation. However, a constant increase in the cell number was observed beginning at 3 days, reaching the pre-cryopreservation level at 5 days post-rewarming. By PNP microplate method, it was found that ALP activity reduced at 1 and 4days post-rewarming, but compared with the level of pre-cryopreservation there were no significant difference. However, a constant increase in ALP activity was detected since 4 days. By real-time PCR, osteogenic gene expression including Runx2, OCN, ALP, COL-1 reduced at days 1 and 4, but compared with the level of pre-cryopreservation there was no significant difference. However, a constant increase in the osteogenic gene expression was since 4 days. The cell viability and osteogenic function were observed without significant difference at each time point after rewarming of cells that had undergone vitreous cryopreservation for 1 or 12 weeks. Preliminary findings indicate that vitreous cryopreservation can maintain cellular viability and osteogenic function of tissue-engineered bone. Cryopreservation time (1 and 12 weeks) has no significant effect on the cell viability and osteogenic function of the tissue-engineered bone after rewarming.